Two-photon microscope with spectral resolution

ABSTRACT

A microscope for generating an image of a sample, the microscope includes a light source for generating a pulsed light; a scanning mirror receiving the pulsed light and transmitting the pulsed light to the sample to be imaged causing the sample to emit energy; a dispersive element that receives the emitted energy from the sample, disburses the energy into its spectral elements and transmits the spectrally disbursed energy; and a camera that generates a spectrally resolved image of the sample based on the spectrally disbursed energy from the dispersive element. Also described is a method of generating spectrally resolved images of the sample.

RELATED APPLICATIONS

The present patent application claims priority to U.S. Provisional Patent Application Ser. No. 60/848,351 filed Sep. 29, 2006, the entire contents of which are herein incorporated by reference.

BACKGROUND

Laser scanning microscopes (such as two-photon and confocal microscopes) are widely used for acquiring images of narrow sections of cells and tissues, in which molecules of interest are tagged with fluorescent molecules, so that the latter render the former visible through emission of light. By acquiring multiple images of such thin sections, three-dimensional images of the samples can be obtained. In experiments involving fluorescence tagging with multiple colors, such as in studies of protein co-localization or protein-protein interactions (studied through Fluorescence Resonance Energy Transfer—FRET—which involves at least two different tags), a fourth dimension becomes necessary, i.e., the spectral dimension (i.e., the wavelength of the emitted light).

SUMMARY

Various detection schemes for spectral properties have been implemented in most commercial confocal microscopes. However, confocal microscopes may suffer from some or all of the following problems, when compared to two-photon microscopes: (1) low acquisition speeds, mostly due to use of a point-scan method; (2) photodestruction (bleaching) of fluorescent light (while the signal is only read from a thin layer), and long time necessary to excite the sample, due to slow reading of the multiple wavelengths at each point; and (3) low signal-to-noise ratio, due to the fact that excitation and emission happen at wavelengths close to one another, making filtering of the signal difficult. On the other hand, known two-photon microscopes, which avoid problems (2) and (3) above, either do not present spectral resolution at all, or, when they do, they suffer from slow acquisition speed.

In one form, the invention provides a microscope for generating an image of a sample, the microscope comprising: a light source for generating a pulsed light; a scanning mirror receiving the pulsed light and transmitting the pulsed light to the sample to be imaged causing the sample to emit energy; a dispersive element that receives the emitted energy from the sample, disburses the energy into its spectral elements and transmits the spectrally disbursed energy; and a camera that generates a spectrally resolved image of the sample based on the spectrally disbursed energy from the dispersive element

The invention also provides a method of generating an image of a sample having x and y dimensions and using a microscope having a laser light source, a computer controlled scanning mirror, a dispersive element and a camera, the method comprising: generating laser energy; scanning the laser energy across the sample to cause emission of light from the sample; disbursing the light emitted from the sample into its spectral elements; and generating a spectrally resolved image of the sample at a given wave length.

The present invention seeks to produce spectrally resolved fluorescence images of samples using a two-photon microscope after only one full scan of the sample, and avoid all of the problems mentioned above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a microscope 10 embodying the invention.

FIG. 2 is a schematic representation of another microscope 100 embodying the invention.

FIG. 3 is a schematic representation of yet another microscope 200 embodying the invention.

FIG. 4 is a schematic representation of yet another microscope 300 embodying the invention.

FIG. 5 is a series of plots generated in a calibration procedure and showing the relationship between rows of image pixels and the corresponding spectral dimension (wavelength).

FIG. 6 illustrates the procedure for reconstructing an image of the fluorescence emission of the sample at a wavelength of interest.

FIG. 7 is a series of spectrally-resolved images acquired using the microscope shown in FIG. 1.

DETAILED DESCRIPTION

Before any embodiments of the invention are explained in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the following drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless specified or limited otherwise, the terms “mounted,” “connected,” “supported,” and “coupled” and variations thereof are used broadly and encompass both direct and indirect mountings, connections, supports, and couplings. Further, “connected” and “coupled” are not restricted to physical or mechanical connections or couplings.

FIG. 1 shows one form of a two-photon microscope 10 with spectral resolution embodying the present invention. The microscope 10 includes a high power solid state laser 12. In one embodiment, the laser 12 is a Verdi brand laser produced by Coherent Inc, and operating at five watts to provide continuous wave (CW) light at a wavelength of 532 nanometers (nm). Other embodiments can employ different lasers operating in different formats as appropriate. The light generated from laser 12 is used to pump a modelocked Ti:Sapphire laser 14. In one embodiment, the Ti:Sapphire laser 14 is a Kapteyn-Murnane Labs laser, of course, other suitable brands may be substituted. The Ti:Sapphire laser 14 generates femtosecond pulses of near-infrared light (centered at approximately 800 nm with a bandwidth of 120 nm). The output of the laser 14 is steered by fixed mirror 16 as well as two computer-controlled, x-y scanning mirrors 18 (x and y here refer to two mutually orthogonal directions on the sample and the corresponding directions on the detector 34). In one embodiment the computer-controlled, x-y scanning mirrors 18 are a pair of mirrors attached to galvanometric scanners (10 mm aperture) that are produced by Nutfiled Technology. The beam of light is then expanded by telescope 20, and passed to a short-pass dichroic mirror 22. Telescope 20 has a conventional round lens which focuses the beam to a point. Short-pass dichroic mirror 22 reflects the light in the focused beam with a long wavelength, and transmits the light in the beam having a shorter wavelength (in this embodiment, light in the visible spectrum). The beam of light is then focused to a diffraction-limited spot on the sample to be imaged 24 by an infinity-corrected high numerical aperture objective 26.

As the diffraction-limited spot is scanned across the sample 24 in the x-direction by the x-y scanning mirrors 18, the back-propagating fluorescence emission from the sample is collected by the objective 26, sent through the short-pass dichroic mirror 22, the tube lens 28, the short-pass filter 30, and is dispersed into spectral components by a light dispersive element 32, in this embodiment, an optical grating. The dispersed beam is projected onto the two-dimensional charge coupled device (“CCD”) detector 34. The terms detector, CCD detector, camera and CCD camera are used interchangeably herein. The short-pass dichroic mirror 22 allows visible light to pass through while reflecting most of the infrared light. The short-pass filter 30 rejects any residual infrared component in the back-propagating light, which would otherwise overwhelm the visible components of interest. The spectral components of the fluorescence emission from the scanned spot are dispersed by the optical grating 32 to form a line on the CCD detector 34 corresponding to each scanned spot (or x position on the sample). In one embodiment of the invention (not shown), a transmission grating is used as the dispersive element 32, but a reflection grating can also be used as the dispersive element 32. Also, in other embodiments (also not shown), it is possible to use a prism as dispersive element 32, as well as any other spectral element capable of dispersing the spectral components of the fluorescence emission from the scanned spot.

Using scanning mirrors 18, the laser light is directed across the sample (along, for example, the x-direction) such that the fluorescence emission spectrum sweeps across the detector 34 to form a rectangle. One dimension of this rectangle corresponds to the scanning direction, with the emission spectrum at each point spread out in the other dimension. In other words, the x-direction on the detector 34 still corresponds to the x-direction on the sample, but the y-direction on the detector 34 corresponds to the spectral dimension (i.e., wavelength) of the sample rather than the y-direction of the sample. One two-dimensional image is captured for each line scanned on the sample in which the x-dimension corresponds to the actual x-dimension of the sample, and the y-dimension corresponds to the spectral dimension (wavelength). Scanning the laser across the sample in the x-direction creates a full image of the spectral components of the fluorescence emission for that y-position. A computer (not shown) is connected to the ccd camera, and the full spectral image is stored in the computer for each position in the y-direction. After performing line scans for different y-values of the sample (through control of the x-y scanning mirrors 18), and storing the images in the computer, the resultant images are reconstructed (as described below) to give the final spectrally resolved fluorescence images.

FIGS. 2 and 3 illustrate two additional embodiments of the invention 100 and 200, respectively. Like parts are identified using like reference numerals. As shown in FIG. 2, the microscope 100 includes a short-pass, dichroic mirror 102 used as one of the computer-controlled, scanning mirrors 18. This results in a half-descanned detection. That is, the back-propagating light is made immobile in the direction controlled by the top scanning mirror 104. FIG. 3 shows yet another microscope 200 embodying the invention and employing half-descanned detection. Microscope 200 includes a long-pass dichroic mirror 202 is inserted between the two scanning mirrors 18. Long-pass dichroic mirror 202 is used here so that only the visible components of the back-propagating light are sent to the detector 34. Similar to the design shown in FIG. 2, the back-propagating light is made immobile in the direction controlled by the top scanning mirror

FIG. 4 illustrates another microscope 300 embodying the invention. Again, like parts are identified using like reference numerals. As shown in FIG. 4, a cylindrical lens 302 is used in telescope 20, so that the focused beam is a line (e.g., along the x-axis of the sample), instead of a point. The line lying along the x-axis is scanned by a single scanning mirror 304 in the y-direction. The emission spectra for each sample point across the line remain aligned along the y-axis and are acquired simultaneously by the CCD detector 34 in a single run. In this way, the time necessary for the other designs to scan the focal point along the x-direction (which involves repeated communication with a computer) is reduced to the time it typically takes to acquire a single point. This leads to a significant increase in the acquisition speed.

The reconstruction procedure (which is performed by the computer) is illustrated in FIGS. 5 and 6. To obtain the fluorescence emission image for a particular wavelength, let us say, λ₅, one finds the row number on the first image (i.e., the spectrally resolved image corresponding to the first y-position on the sample) that corresponds to the desired wavelength (row five in this example). The desired wavelength may be chosen based on knowledge of the particular wavelength at which the sample fluoresces. Alternatively, the images can be spectrally resolved at all wavelengths if there is insufficient knowledge to target a specific wavelength. Still referring to FIG. 5, the next row of the next image (i.e., row six on the second image) would, by virtue of the calibration procedure, correspond to the same wavelength but correspond to the next y-value, and so on. Stacking all the image rows that correspond to the wavelength of interest, an image of the fluorescence emission of the sample at the wavelength of interest is obtained. This procedure is then repeated for other wavelengths. Spectrally-resolved images acquired with the preferred implementation of the invention are shown in FIG. 7. These images show yeast cells expressing a membrane receptor (Sterile 2-alpha factor protein (“Ste2p”)) tagged with a fluorescent tag (Green Fluorescent Protein (“GFP”)).

By using a standard sample that fluoresces uniformly across the sample (e.g., fluorescein), the line-scanning procedure can be calibrated so that from line-scanning at one y-value to the next on the sample, let us say, from y=y₀ to y=y₀+Δy, the fluorescence spectrum moves by exactly one pixel along the spectral dimension on the detector 34. This calibration procedure also allows identification of the relation between row numbers and wavelengths on each of the images as shown in FIG. 5. When the back-propagating light is made immobile in the y-direction (such as with the microscopes 100 and 200), the fluorescence spectra will be immobile on the detector. In this case, the reconstruction procedure is very straightforward, since a particular row always corresponds to the same wavelength in all the images and so no calibration is required. In these embodiments, a dichroic mirror replaces the scanning mirror or is positioned in the narrow space between the scanning mirrors.

In any of the embodiments described, one full scan of the sample gives spectrally resolved fluorescence images of the sample. This can lead to a tremendous increase in acquisition speed when fast detectors 34 are used. All of the embodiments also allow for transmission imaging. The only modification required is to provide a broadband light source beneath the sample stage and a narrow band-pass filter (such as an interference filter) in the optical path somewhere between the sample and the detector 34. Without the band-pass filter, multiple “ghost” images of the sample may be projected onto the detector 34 due to the grating. The band-pass filter removes all but one of these images.

The embodiments described herein provide for much faster acquisition of spectrally resolved images than known microscopes. Specifically, the acquisition speed for the point-scan configuration can be, theoretically, at least fifty times higher than that of confocal microscopes. Speeds of about three times higher (thirty seconds compared to ninety seconds) than that of a Leica confocal microscope have been achieved while acquiring images at ten times the number of distinct wavelengths (i.e., ten times higher spectral resolution). In addition, the line-scan design should allow a much higher speed, theoretically at least a factor of five times better than the point-scan (i.e., close to video-rate acquisition speed), due to removal of the x-scanner, the speed of which is limited by its mechanics and the necessity of communicating with the computer controlling the scanner. Moreover, the microscopes described also allow for transmission imaging.

Various features and advantages of the invention are set forth in the following claims. 

1. A microscope for generating an image of a sample, the microscope comprising: a light source for generating a pulsed light; a scanning mirror receiving the pulsed light and transmitting the pulsed light to the sample to be imaged, causing the sample to emit energy; a dispersive element that receives the emitted energy from the sample, disburses the energy into its spectral elements and transmits the spectrally disbursed energy; and a camera that generates a spectrally resolved image of the sample based on the spectrally disbursed energy from the dispersive element.
 2. A microscope as set forth in claim 1 wherein the light source comprises a laser.
 3. A microscope as set forth in claim 1 wherein the scanning mirror comprises a computer controlled x-y scanning mirror.
 4. A microscope as set forth in claim 1 wherein the scanning mirror comprises a plurality of computer controlled mirrors, and wherein one of the mirrors is a short pass, dichroic mirror.
 5. A microscope as set forth in claim 1 wherein the scanning mirror comprises a pair of computer controlled scanning mirrors and a long pass dichroic mirror positioned between the computer controlled scanning mirrors.
 6. A microscope as set forth in claim 1 wherein the dispersive element comprises one of an optical grating, transmission grating, reflection grating, or prism.
 7. A microscope as set forth in claim 1 wherein the camera comprises a charge coupled device camera.
 8. A microscope as set forth in claim 1, and further comprising a round lens for focusing the light from the light source to a point.
 9. A microscope as set forth in claim 1, and further comprising a cylindrical lens for focusing the light from the light source in a line.
 10. A microscope as set forth in claim 1, and further comprising a broadband light source positioned beneath the sample to be imaged and a narrow band-pass filter positioned between the sample and the camera to provide transmission imaging of the sample.
 11. A microscope for generating an image of a sample, the microscope comprising: a laser light source for generating a pulsed light; a computer controlled, x-y scanning mirror receiving the pulsed light and transmitting the pulsed light to the sample to be imaged, causing the sample to emit energy; at least one of an optical grating, transmission grating, reflection grating, or prism that receives the emitted energy from the sample, disburses the energy into its spectral elements and transmits the spectrally disbursed energy; and a charge coupled device camera that generates a spectrally resolved image of the sample based on the spectrally disbursed energy from the dispersive element.
 12. A method of generating an image of a sample having x and y dimensions and using a microscope having a laser light source, a computer controlled scanning mirror, a dispersive element and a camera, the method comprising: generating laser energy; scanning the laser energy across the sample to cause emission of light from the sample; disbursing the light emitted from the sample into its spectral elements; and generating a spectrally resolved image of the sample at a given wave length.
 13. A method as set forth in claim 12 wherein disbursing the light from the sample into its spectral elements comprises passing the light through the dispersive element.
 14. A method as set forth in claim 13 wherein the dispersive element is a one of an optical grating, transmission grating, reflection grating, or prism.
 15. A method as set forth in claim 12 wherein generating a spectrally resolved image of the sample at a given wave length comprises acquiring a series of spectral images for each y-position on the sample and reconstructing an image from the components of each of the series of spectral images corresponding to the given wave length.
 16. A method as set forth in claim 12 and further comprising calibrating the microscope by generating fluorescence images of a sample that fluoresces uniformly across the sample and relating row numbers in the y direction with spectral elements of the emitted light from the sample.
 17. A method of generating an image of a sample having x and y dimensions and using a microscope having a laser light source, a computer controlled scanning mirror, a dispersive element and a camera, the method comprising: generating laser energy; scanning the laser energy across the sample to cause emission of light from the sample; disbursing the light emitted from the sample into its spectral elements by passing the light through a dispersive element such as an optical grating, a transmission grating, a reflection grating, or a prism; and generating a spectrally resolved image of the sample at a given wave length by acquiring a series of spectral images for each y-position on the sample and reconstructing an image from the components of each of the series of spectral images corresponding to the given wave length. 